Protein kinases play a critical role in the regulation of virtually all aspects of cellular regulation and comprise one of the most active areas of research in the pharmaceutical industry today. The 522 protein kinase domains in the human genome may provide tremendous opportunities for developing new drugs for untreated disease and the development of protein kinase inhibitors has increasingly become a major focus for the pharmaceutical industry. Protein kinase inhibitors have been reported to be useful in the treatment of numerous diseases including cancer, inflammatory and immunological diseases. See for example I. K. Mellinghoff and C. L. Sawyers, Kinase Inhibitor Therapy in Cancer, 14(12):1–11, 2000; J. Dumas, Growth factor receptor kinase inhibitors: recent progress and clinical impact, Current Opinion in Drug Discovery & Development, 4(4):378–89, 2001; J. Dumas, Protein kinase inhibitors: emerging pharmacophores, 1997–2000, Expert Opinion on Therapeutic Patents. 11(3):405–429, 2001; D. H. Williams and T. Mitchell, Latest developments in crystallography and structure-based design of protein kinase inhibitors as drug candidates, Current Opinion in Pharmacology, 2(5):567–73, 2002; S. B. Noonberg and C. C. Benz, Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents, Drugs, 59(4):753–67, 2000; S. Brunelleschi , L. Penengo, M. M. Santoro and G. Gaudino, Receptor tyrosine kinases as target for anti-cancer therapy, Current Pharmaceutical Design. 8(22):1959–72, 2002; P. G. Goekjian and M. R. Jirousek, Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development, Current Medicinal Chemistry. 6(9):877–903, 1999, A. Gordon, The increasing efficacy of breast cancer treatment, Clinical Oncology (Royal College of Radiologists), 9(5):338–42, 1997.
While this large gene family represents a rich source on new drug targets, developing assays used to determine compound affinity is highly problematic. Current high throughput screening assays for protein kinase inhibitors measure the incorporation of phosphate into a protein or peptide substrate. The most established method for assaying protein kinase inhibitors is a radiometric assay in which the gamma phosphate of ATP is labeled with either 32P or 33P. When the kinase transfers the gamma phosphate to the hydroxyl of the protein substrate during the phosphor-transferase reaction the protein becomes covalently labeled with the isotope. The protein is removed from the labeled ATP and the amount of radioactive protein is determined. This assay is still the gold standard for quantitative protein kinase assays. Adaptation of this assay into a high throughput format is problematic due to the labor intensive separation steps and the large amounts of radioactivity that are used.
An alternative radiometric assay that is capable of higher throughput is the SPA or scintillation proximity assay (Amersham International). In this assay scintillant impregnated beads emit light when the labeled substrate is bound to the bead. This assay is limited by the level of radioactivity and the efficiency of the peptide substrate.
Most non-radioactive assays use antibodies that recognize the product of the kinase reaction, i.e. a phospho-peptide. The binding assays use antibodies detected with enzyme-catalyzed luminescent readout. These methods are limited by reagent availability, well coating, and multiple wash and incubation steps.
Techniques using fluorescence polarization to measure either protein kinase activity or inhibitor binding rely on a labeled antibody or peptide substrate. In these assays the enzyme transfers the gamma phosphate of ATP to a protein or peptide substrate. This activity is monitored by detecting the phosphor-peptide by such means as an antibody. The binding of the antibody to the phosphor-peptide will slow the free rotation of the peptide in solution and, therefore, a polarization signal from the product of the catalytic reaction can be detected. Examples include Burke et al. US 2001/0004522 A1 or T. C. Turek et al., Analytical Biochemistry, 2001, 299 (1), 25–53.
Each of the assays described above determines the affinity of an inhibitor based on reductions in the enzyme's product formation. Despite many alternatives to measure enzyme product, each assay format requires an active enzyme, a high affinity substrate and a specialized antibody to bind to the phospho-peptide. In most cases, obtaining an active enzyme involves phosphorylation of the activation loop which lies across the catalytic cleft. This phosphorylation can be highly problematic when the upstream kinase performing this function is unknown. Even if the activating kinase is known, the requirement necessitates cloning or co-expressing multiple kinases. In some cases, additional cofactors such as the cyclins must be added to fully activate the protein.
Protein kinases display a great deal of sequence specific substrate selection. Therefore, suitable specific substrate must be found for each kinase. In addition, if the substrate site is a serine or threonine then specialized phosphor-specific antibodies must be obtained to monitor activity in a high throughput assay format. These requirements add uncertainty to the design of new kinase assays as well as added expense.
In summary, problems with protein kinase assay development are most often encountered with the activation step and in protein substrate selection. Development of a high throughput screening assay for new protein kinases currently takes 6–12 months. In addition to cloning and expressing the protein the enzyme must be activated, substrates must be found, and reagents to detect the product of the enzyme reaction must be generated. This is a very time consuming and labor intensive effort and each assay must be specialized for one or a small subset of the protein kinases.
Any assay that employs methods to bind phospho-peptide substrate is limited by the concentration of ATP used in the assay. This is inevitable as both the phosphorylated amino acids of the peptide substrate and the phosphates on ATP compete with each other for the binding site on the added reagent. Kinases with medium to high Km values assayed at sub Km values make substrate conversion problematic.
Finally, it is desirable to have an assay that expresses compound affinity as Kd rather than IC50. This allows for transparent comparison of inhibitors between kinases, critical for determining compound selectivity. One cannot compare IC50 measurements from kinase to kinase without additional ATP Km data.
To overcome these problems it would be desirable to have an assay to measure inhibitor affinities that is compatible with high throughput screening, is homogeneous (no wash steps), does not require activated protein, does not require a catalytic reaction, is substrate independent, is not dependent on ATP concentrations and measures compound Kd values. Such an assay would save institutes and companies involved in drug discovery research millions of dollars by reducing assay development time and the cost of reagents.